Development and evaluation of multifunctional agents for potential treatment of Alzheimer's disease: Application to a pyrimidine-2,4-diamine template

Article abstract of DOI:10.1016/j.bmcl.2012.05.077

We investigated a group of 2-benzylpiperidin-N-benzylpyrimidin-4-amines with various electron-withdrawing or electron-donating groups (EWGs or EDGs, respectively) as multi-targeted Alzheimer's disease (AD) therapeutics. The synthesized derivatives were sc

Full text of DOI:10.1016/j.bmcl.2012.05.077

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4707–4712
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development and evaluation of multifunctional agents for potential treatment
of Alzheimer’s disease: Application to a pyrimidine-2,4-diamine template
Tarek Mohamed a,b, Jacky C. K. Yeung b, Maryam S. Vasefi a,c, Michael A. Beazely a, Praveen P. N. Rao a,
⇑
a School of Pharmacy, Health Sciences Campus, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1
b Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1
c Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1
a r t i c l e i n f o
a b s t r a c t
Article history:
We investigated a group of 2-benzylpiperidin-N-benzylpyrimidin-4-amines with various electron-with-
drawing or electron-donating groups (EWGs or EDGs, respectively) as multi-targeted Alzheimer’s disease
(AD) therapeutics. The synthesized derivatives were screened for anti-cholinesterase (AChE and BuChE),
anti-Ab-aggregation (AChE- and self-induced) and anti-b-secretase (BACE-1) activities in an effort to
identify lead, multifunctional candidates as part of our multi-targeted approach to treat AD. Biological
assessment revealed that the nature of the substituent on the C-4 benzylamine group (e.g., halogen vs
methoxy-based) greatly affected the biological profile. In vitro screening identified N2-(1-benzylpiperi-
din-4-yl)-N4-(3,4-dimethoxybenzyl)pyrimidine-2,4-diamine (7h) as the lead candidate with a dual ChE
Received 5 May 2012
Revised 18 May 2012
Accepted 18 May 2012
Available online 26 May 2012
(AChE IC50 = 9.9
at 100 M) and BACE-1 (34% inhibition at 10
roblastoma cell viability at 40 M = 81.0%). Molecular modeling studies indicate that a central pyrimi-
l
M; BuChE IC50 = 11.4
lM), Ab-aggregation (AChE-induced = 59.3%; self-induced = 17.4%
l
l
M) inhibitory profile along with good cell viability (% neu-
l
dine-2,4-diamine ring serves as a suitable template to develop novel small molecule candidates to
target multiple pathological routes in AD.
Ó 2012 Elsevier Ltd. All rights reserved.
Alzheimer’s disease (AD) is a highly complex and rapidly pro-
gressive, neurodegenerative disease that manifests in the choliner-
gic branch of the central nervous system (CNS).1 The hallmarks of
AD include the rapid onset of cholinergic dysfunction, accelerated
formation and aggregation of amyloid-b (Ab) peptides, collapse of
the neuronal cytoskeleton and formation of neurofibrillary tangles
(NFTs) of hyperphosphorylated tau protein.2–6 Its devastating
pathology is jointly described by the cholinergic, amyloid and tau
hypotheses, respectively, where the end result is neuronal cell
death leading to the onset of dementia symptoms and eventually
death. With an increase in the aging population, AD’s prevalence
amongst the elderly is on the rise.2,3
its actions in the synapse by degrading it to choline and acetate.8,9
A
shift in the ratio of AChE to BuChE has been observed and that
seems to be dependent on the stage of the disease. In early patho-
genesis, AChE is considered the primary degrading enzyme and
therefore, it plays a vital role in disease progression; however, as
neuronal cells are depleted and AChE concentrations fall, BuChE
(the secondary ChE with a wider distribution within the body) acts
as the major degrading enzyme.10,11
The amyloid hypothesis attributes the onset and progression of
AD to the rapid generation and aggregation (metal-ion-promoted,
self-promoted or AChE-promoted) of toxic amyloid-b (Ab) pep-
tides.12–14 Amyloid precursor protein (APP) is normally processed
The cholinergic hypothesis describes AD pathology as a systemic
collapse in cholinergic neurotransmission as a result of the overall
decline in acetylcholine (ACh): a vital neurotransmitter utilized
by sympathetic and parasympathetic neurons.7 Cholinesterase
(ChE isoforms: AChE and BuChE) enzymes act on ACh to terminate
by
b-secretase (BACE-1) takes over generating pro-A b domains
subsequently processed by -secretase, leading to the liberation
a-secretase, but with AD, a shift in APP processing occurs where
c
of Ab1–40/42 peptides.12–14 Recent and ongoing research efforts have
uncovered links between the cholinergic and amyloid hypotheses
including a key role of AChE in promoting Ab-aggregation leading
to highly toxic AChE–Ab peptide complexes as well as the role of
acetycholine receptors (AChRs) in modulating the APP processing
pathways.15–19
Abbreviations: AD, Alzheimer’s disease; ChEIs, cholinesterase inhibitors; hAChE,
human acetylcholinesterase; hBuChE, human butyrylcholinesterase; eqBuChE,
equinebutyrylcholinesterase; SAR, structure–activity relationship; Ab, amyloid-b;
DTNB, 5,50-dithiobis(2-nitrobenzoic acid); ThT, thioflavin T; MTT, 3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyl tetrazolium bromide; BACE-1, b-secretase or beta-site
amyloid precursor protein cleaving enzyme.
Collectively, these complex factors of AD pathology strengthen
the necessity to develop multi-targeted/multifunctional therapies
that could exhibit disease-modifying effects (DMEs) in an effort
to reverse disease manifestation.20
⇑
Corresponding author. Tel.: +1 519 888 4567x21317.
E-mail address: praopera@uwaterloo.ca (P.P.N. Rao).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.077

4708
T. Mohamed et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4707–4712
Currently marketed AD pharmacotherapies ( Fig. 1) such as
AriceptÒ (donepezil–1) and RazadyneÒ (galantamine–2) offer
symptomatic relief, while the latter can be classified as a multi-
functional agent as it targets the ChE enzymes along with enhanc-
ing nicotinic AChR activity and blocking Ab-aggregation.21,22 We
previously reported the development of a group of heterocyclic,
non-fused ChEIs based on a 2,4-disubstituted pyrimidine ring
(2,4-DPR) template, where compounds 3 and 4 (Fig. 1) were iden-
tified as lead derivatives based on their ChE and Ab-aggregation
inhibition profiles.23,24
Here we report the development and evaluation of new hetero-
cycles (7a–j) containing a pyrimidine-2,4-diamine scaffold as novel
small molecules possessing multifunctional activity against the
ChE enzymes, amyloid aggregation (both AChE and self-induced)
and BACE-1.
Scheme 1. Reagents and conditions: (a) DIPEA, primary amines (H2N-R), EtOH, 0 °C
to 85–90 °C and reflux for 4 h. (b) DIPEA, 4-amino-1-benzylpiperidine, n-BuOH,
190–195 °C, 14–16 h.
The synthesis of target pyrimidine-2,4-diamine derivatives was
accomplished in two steps. In the first step, various C-4 substituted
benzyl-2-chloropyrimidin-4-amine intermediates (6a–j) were syn-
thesized from the 2,4-dichloropyrimidine starting material (5) by a
nucleophilic aromatic substitution reaction at C-4 using N,N-diiso-
propylethylamine (DIPEA) and appropriate benzylamine (R1 =
2-chloro, 3-chloro, 4-chloro, 4-bromo, 4-fluoro, 4-methyl, 4-meth-
oxy, 3,4-dimethoxy, 3,4,5-trimethoxybenzylamine and piperonyl-
amine, respectively). The reaction was carried out in EtOH at
80–85 °C and refluxed for 4 h. Intermediates 6a–j were obtained
in moderate to good yield ranging from 60% to 89% (Scheme 1).23–25
In the second step, the C-2 chlorine was displaced by 4-amino-
1-benzylpiperidine. This coupling reaction was run under vigorous
conditions (190–195 °C) for 14–16 h with DIPEA in a sealed pres-
sure vessel (PV) using n-BuOH as a solvent (Scheme 1). The target
pyrimidine-2,4-diamine derivatives (7a–j) were obtained in mod-
erate to good yields (45–54%) (Scheme 1).23,24,26
eqBuChE IC50 = 2.2 to 11.4
tion = inactive to 59.3%; self-induced Ab1–40 aggregation = inactive
to 48.4% and BACE-1 IC50 range = 0.6 to >50 M).
lM; hAChE-induced Ab1–40 aggrega-
l
Based on molecular modeling studies, we incorporated a ben-
zylpiperidine pharmacophore at C-2 position that is present in
the donepezil in order to obtain dual AChE and BuChE inhibition
whereas the C-4-position of the pyrimidine-2,4-diamine was var-
ied to develop a suitable peripheral anionic site (PAS) binding
pharmacophore (7a–j, Table 1). The C-4 halogen-substituted ben-
zyl derivatives (7a–e) exhibited AChE inhibition with IC50 values
ranging from 7.7
ues ranging from 2.2
l
M to 9.9
l
M and BuChE inhibition with IC50 val-
M. The location of a chlorine
lM to 4.1
l
The ability of the synthesized derivatives (7a–j) to inhibit both
ChEs (hAChE and eqBuChE), hAChE-induced and self-induced
Ab1–40 aggregation as well as BACE-1 was evaluated in vitro (IC50
(ortho, meta or para) on the C-4 benzyl substituent (7a, 7b and
7c) did not affect AChE or BuChE inhibition and potency to a great
extent (AChE IC50 = 7.7–8.8
range). Interestingly, the replacement of the p-Cl with a p-F did
not exhibit a significant change either (7e, AChE IC50 = 7.7 M;
BuChE IC50 = 2.2 M) despite the differences in size and electro-
lM range; BuChE IC50 = 2.4–2.8 lM
values or % inhibition at 100 l
M, Tables 1 and 2).23,24,27,28 The
SAR studies indicated that the multifunctional capability of the
pyrimidine-2,4-diamine derivatives was sensitive to steric and
electronic properties at the C-2 and C-4 positions. They derivatives
l
l
negativity. On the other hand, when a bromine halogen is placed
at the para position, it results a slight decrease in AChE potency
and almost twofold decrease in BuChE potency (7d, AChE
exhibited a broad range of inhibition (hAChE IC50 = 7.7 to 12.9 lM;
IC50 = 9.9
pounds from this series exhibited superior BuChE inhibition com-
pared to reference agents donepezil (BuChE IC50 = 3.6 M) and
galantamine (BuChE IC50 = 12.6 M, Table 1).
lM; BuChE IC50 = 4.1 lM) compared to 7e. All com-
l
l
As a next step we explored the possibility of developing a suit-
able pharmacophore at C-4 position of the pyrimidine ring that
could potentially bind to the PAS of AChE.13,17 In this regard, the
C-4 benzyl group was substituted with 3,4-dimethoxy, 3,4,5-
trimethoxy and a fused ring (3,4-methylenedioxy) (7f–j, Table 1).
These compounds were compared with 4-methyl and 4-methoxy-
benzyl substituted counterparts. All compounds from this series
exhibited dual AChE and BuChE inhibition (Table 1). Compound
7g with a C-4 4-methoxybenzyl substituent was the most potent
AChE inhibitor (AChE IC50 = 9.40
ylbenzyl group) was the most potent BuChE inhibitor (BuChE
IC50 = 2.5 M, SI = 5.2). All the derivatives (except 7h and 7i) exhib-
ited potent BuChE inhibition (IC50 = 2.2 M to 4.9 M) similar to
that of donepezil (IC50 = 3.6 M) and surpassing that of galanta-
mine (IC50 = 12.6 M).
lM) whereas 7f (with a C-4 p-tol-
l
l
l
l
l
Among 7a–j series, 7e with a C-4 4-fluorobenzyl substituent
was identified as a lead molecule with good AChE/BuChE inhibition
and potency (AChE IC50 = 7.7 lM, BuChE IC50 = 2.2 lM, Table 1).
The ability of a number of pyrimidine-2,4-diamine derivatives
to inhibit hAChE-induced or self-induced Ab1–40 aggregation was
evaluated by a thioflavin T (ThT) fluorescence method and the
Fig. 1. Structures of some anti-AD agents (1–4).

T. Mohamed et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4707–4712
4709
Table 1
AChE and BuChE inhibitory activities of pyrimidine-2,4-diamine derivatives (7a–j) and their ClogP data along with molecular volumes (MVs)
a
Compd
EWGs
R1
IC50
(
lM)
SIb
ClogPc
MVd
AChE
BuChE
7a
7b
7c
7d
7e
2-Cl
3-Cl
4-Cl
4-Br
4-F
7.70 0.77
7.70 0.77
8.80 0.88
9.90 1.00
7.70 0.70
2.40 0.24
2.50 0.23
2.80 0.28
4.10 0.40
2.20 0.20
3.2
3.1
3.1
2.4
3.5
5.32
5.32
5.32
5.48
4.74
277.8
277.8
277.8
287.1
263.1
EDGs
7f
7g
7h
7i
4-Me
4-OMe
3,4-diOMe
3,4,5-triOMe
3,4-[Dioxole]
12.90 0.12
9.40 0.90
9.90 0.99
10.30 0.10
12.60 0.12
2.50 0.22
4.90 0.50
11.40 0.10
7.70 0.65
3.90 0.40
5.2
1.9
0.9
1.3
3.2
5.10
4.52
4.26
3.90
4.57
272.7
278.8
299.4
319.3
280.9
7j
Donepezil (1)
Galantamine (2)
—
—
0.032 0.003
3.20 0.32
3.60 0.32
12.60 0.11
—
0.3
4.60
1.0
271.0
179.2
a
The in vitro test compound concentration required to produce 50% inhibition of hAChE and eqBuChE. The result (IC50) is the mean of two separate experiments (n = 4) and
the deviation from the mean is <10% of the mean value.
b
Selectivity Index = hAChE/eqBuChE IC50
ClogP was determined using ChemDraw Ultra 12.0. CambridgeSoft Company.
Molecular volumes (MV) were calculated after a minimization protocol using the molecular properties calculator in the Discovery Studio program from Accelrys Inc
.
c
d
(San Diego, CA).
results were compared with reference compounds propidium io-
amyloid fibrils (pre-fibrillization, PF assay, Fig. 1, Supplementary
data). The TEM results confirmed the ability of compounds 7c (Pa-
nel B, Fig. 1, Supplementary data) and 7d (Panel D, Fig. 1, Supple-
mentary data) to prevent the formation of amyloid fibrils when
co-incubated with Ab1–40 peptide compared to control incubation
(Panel A, Ab1–40 alone). More significantly, they were also able to
dissolve preformed amyloid fibrils when incubated at a concentra-
dide and donepezil (Table 2).29 At a concentration of 100
lM, they
exhibited varying degrees of inhibition (inactive to 59% inhibition
of aggregation). Generally pyrimidine-2,4-diamine derivatives
exhibited inhibition of both AChE-induced and self-induced of
Ab1–40 aggregation (7a–j, Table 2).
In the hAChE-induced assay, inhibition ranged from inactive (7a
and 7c) up to 59% (7h) while in the self-induced assay, inhibition
ranged from inactive (7i) up to 48% (7d). Interestingly, compounds
that possessed C-4 EWG group (7a–e; Cl, Br or F, respectively) were
more effective in the self-induced assay while compounds that
possessed C-4 EDGs (7f–j; Me, 4-OMe, 3,4-diOMe, 3,4,5-triOMe
and 3,4-methylenedioxybenzyl) substituted derivatives were more
effective in AChE-induced assay. Among these, derivative 7h was
identified as the most effective inhibitor of AChE-induced aggrega-
tion (59.3% inhibition, Table 2) and was ꢀ1.3-fold more potent
compared to galantamine (48% inhibition). It is plausible that the
presence of a C-2 amino-1-benzylpiperidine substituent might
play a role in orienting the C-4 3,4-dimethoxybenzyl substituent
toward PAS of AChE. It is noteworthy that 7d (4-bromobenzyl sub-
stituent at C-4 position) exhibited dual mode aggregation inhibi-
tion in both hAChE and self-induced assay (53.90% and 48.40%
inhibition, respectively, Table 2).
tion of 10 lM (Panels C and E, respectively, Fig. 1, Supplementary
data). Over the course of a 6-day co-incubation experiment, both
7c and 7d demonstrated anti-aggregation properties similar to that
of galantamine (Panel F, Fig. 1, Supplementary data). A three day
pre-fibrillization assay prior to test compound incubation both 7c
(Panel C) and 7d (Panel E) demonstrated their ability to dissolve
preformed amyloid fibrils with 7d exhibiting greater degree of
amyloid fibril disassembly relative to 7c.
The aspartyl protease enzyme BACE-1 is involved in the forma-
tion of amyloid-b by cleaving the amyloid precursor protein
(APP).29–31 Recent work from Merck scientists has shown that
pyrimidine ring templates could be useful to design potent BACE
inhibitors.31 Since our small molecules are based on a pyrimidine
ring scaffold we investigated the ability of BP-derivatives (7a–j)
to inhibit BACE-1 and results were compared with reference com-
pounds donepezil and the peptide inhibitor N-benzyloxycarbonyl-
Val-Leu-Leucinal (Table 2).
Lead derivatives from the self-induced assay (7c and 7d, 43.6%
and 48.4% inhibition, respectively at 100
l
M), were further exam-
Activity against b-secretase (IC50 values) ranged from 0.6 to
ined using transmission electron microscopy (TEM) to assess their
anti-aggregation activity/morphology in the presence of amyloid
peptides (co-incubation, CI assay) and in the presence of preformed
>50 lM and was greatly influenced by steric and electronic proper-
ties. Derivatives with a C-2 benzylpiperidine pharmacophore gen-
erally exhibited potent activity with the exception of 7f (a p-Me

4710
T. Mohamed et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4707–4712
Table 2
Anti-amyloid and BACE-1 inhibition activities of 7a–j
R1
% Inhibition at 100
l
Ma
BACE-1 IC50 (lM)
b
Compd
EWGs
hAChE-induced
Self-induced
7a
7b
7c
7d
7e
2-Cl
3-Cl
4-Cl
4-Br
4-F
NA
42.20 0.40
39.90 0.35
42.60 0.39
48.40 0.46
29.80 0.30
1.70 0.15
0.60 0.05
3.20 0.30
33%e
27.20 0.20
NA
53.90 0.45
38.10 0.33
0.70 0.05
EDGs
7f
7g
7h
7i
4-Me
4-OMe
3,4-diOMe
3,4,5-triOMe
45.10 0.45
36.20 0.30
59.30 0.48
32.70 0.30
50.20 0.50
17.00c
21.90 0.20
18.20 0.20
17.40 0.15
NA
11.10 0.10
0.60 0.04
34%e
8.90 0.90
1.40 0.13
3.20 0.29
ND
7j
3,4-[Dioxole]
18.60 0.20
ND
Donepezil (1)
Galantamine (2)
Propidium
—
—
—
ND
82.00
48.00d
ND
ND
N-Benzyloxycarbonyl-Val-Leu-Leucinal
ND
ND
14.00 0.15
a
The result (% aggregation inhibition of Ab1–40 at 100 lM) is the mean of two separate experiments (n = 4) and the deviation from the mean is <10% of the mean value.
The result (IC50) is the mean of duplicate readings (n = 2–4) and the deviation from the mean is <10% of the mean value.
Previously reported (Ref. 23).
Ref. 21. NA = Not active. ND = Not determined.
b
c
d
e
% BACE-1 inhibition at 10 lM.
benzylamine at C-4) and 7i (a tri-OMe benzylamine at C-4). When
compared to a peptidic BACE-1 inhibitor (N-benzyloxycarbonyl-
tyrosine hydroxyl groups and the C-2 NH and pyrimidine N-3 (dis-
tance <3.5 Å). The C-4 group (4-fluorobenzylamine) was oriented
such that the fluorine atom was undergoing hydrogen bonding
interactions (Asp74 and Asn87; distance ꢀ3.5 Å). This orientation
Val-Leu-Leucinal, IC50 = 14.0
and 7g (p-OMe) exhibited ꢀ22-fold increase in potency
(IC50 = 0.6–0.7 M). These studies indicate that a pyrimidine-2.4-
lM), derivatives 7b (m-Cl), 7e (p-F)
l
was assisted by Trp86 that was undergoing p–p interactions with
diamine based non-peptidic molecules can target BACE-1.
The cytotoxicity of the lead derivative 7h with multifunctional
activities (dual ChE, AChE-induced amyloid aggregation and
BACE-1 inhibition) possessing a C-4 3,4-dimethoxybenzyl PAS
pharmacophore was evaluated using 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl tetrazolium bromide (MTT) based colorimetric as-
say. Compound 7h exhibited good cell viability (81%) suggesting
that a pyrimidine-2,4-diamine scaffold serves as a suitable tem-
plate to develop small molecule candidates as multi-targeting
agents to treat AD. Furthermore pyrimidine-2,4-diamines (7a–j)
exhibit ClogP values from 3.90 to 5.48. In this regard, compound
7h exhibited a ClogP value of 4.26 which compares well with the
anti-AD reference agent donepezil (ClogP = 4.60, Table 1) indicat-
ing its potential to reach the CNS.
the C-4 phenyl ring (distance ꢀ4 Å). The C-2 benzylpiperidine
pharmacophore was oriented in a region comprised of Trp286,
Tyr341 and Tyr337. Interestingly, the benzyl substituent was posi-
tioned at the posterior face of Trp286 (PAS). Overall compound
exhibited a linear conformation spanning the catalytic active site
(CAS) and PAS which explains its ability to bind to AChE.
Furthermore, the most potent BuChE inhibitor 7e [N2-(1-ben-
zylpiperidin-4-yl)-N4-(4-fluorobenzyl)pyrimidine-2,4-diamine]
(eqBuChE IC50 = 2.2 lM) was investigated by docking studies with
hBuChE (Fig. 3). The most stable enzyme–ligand complex indicates
that the pyrimidine-2,4-diamine template was located at the cen-
ter of the active site gorge (ꢀ10 Å away from the catalytic triad
His447 residue at the bottom of the active site and ꢀ12 Å away
from the entry site residue, Ala277). Similar to its hAChE binding
mode, the entire C-4 group (4-fluorobenzylamine) was oriented
such that the fluorine atom toward His438 (hydrogen bonding
interaction; distance ꢀ3.5 Å) and Ser198. The C-2 benzylpiperidine
pharmacophore was oriented in a hydrophobic region alongside
Pro285 and acyl pocket (Leu286, Ser287, Val288; distances ꢀ4–
5 Å) with the phenyl ring was about ꢀ4 Å away from Trp231 and
Phe398. It was interesting to note that 7e (molecular volume
263.1 Å3) was oriented in an inverse U-shaped conformation and
was able to interact with key residues within the active site of
BuChE.
The binding interactions of potent AChE and BuChE inhibitors
were investigated by molecular modeling (docking). The most
stable enzyme–ligand complex for compound 7e [N2-(1-benzylpi-
peridin-4-yl)-N4-(4-fluorobenzyl)pyrimidine-2,4-diamine; hAChE
IC50 = 7.7 lM) in hAChE] (Fig. 2) indicated that the pyrimidine-
2,4-diamine template was located at the center of active site gorge
(ꢀ6.5 Å away from the catalytic triad His447 residue at the bottom
of the active site and ꢀ7 Å away from the PAS Trp286). The
pyrimidine ring was also oriented between Tyr124 and Tyr337
and was undergoing hydrogen bonding interactions with the

T. Mohamed et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4707–4712
4711
Figure 3. The binding mode of N2-(1-benzylpiperidin-4-yl)-N4-(4-fluoroben-
zyl)pyrimidine-2,4-diamine (7e, ball and stick) in hBuChE (PDB: 1P0I). Red dotted
lines indicate hydrogen bonding interactions. Hydrogen atoms are removed to
increase clarity.
Figure 2. The binding mode of N2-(1-benzylpiperidin-4-yl)-N4-(4-fluoroben-
zyl)pyrimidine-2,4-diamine (7e, ball and stick) in hAChE (PDB: 1B41). Red dotted
lines indicate hydrogen bonding interactions. Hydrogen atoms are removed to
increase clarity.
inhibition at 10 lM) with good cell viability in the SH-SY5Y
neuroblastoma cell line (81%).35,36 Together, these results imply that
pyrimidin-2,4-diamines are suitable ring templates to design
anti-AD small molecule therapeutics as disease-modifying agents
(DMAs).
In order to understand the binding mode of compound 7h
[N2-(1-benzylpiperidin-4-yl)-N4-(3,4-dimethoxybenzyl)pyrimidine-
2,4-diamine] that exhibited good inhibition of AChE-induced
Ab-aggregation (59% inhibition, Table 2), it was docked within the
active site of hAChE and was compared with the binding mode of
reference agent donepezil that is known to orient the 3,4-dimethoxy
group closer to AChE–PAS (Fig. 4). It should be noted that the com-
pound 7h (molecular volume = 299.4 Å3, Table 1) is much larger as
compared to donepezil (molecular volume = 271 Å3). This investiga-
tion shows that the C-4 3,4-dimethoxybenzylamine substituent of
7h was much closer to the PAS residues (distance ꢀ4 Å) compared
to donepezil and was tightly stacked against the indole ring of
Trp286 correlating with its superior activity against AChE-induced
Acknowledgments
The authors would like to thank Faculty of Science and the
School of Pharmacy at the University of Waterloo for financial sup-
port of this research project.
Ab-aggregation compared to donepezil (59% vs 17% at 100 lM). In
this regard, a C-4 3,4-dimethoxybenzyl substituent could be consid-
ered as a suitable PAS binding pharmacophore in the development
of novel pyrimidin-2,4-diamine based small molecules.
The SAR data obtained from this pyrimidin-2,4-diamine chemical
library indicates that steric and electronic properties at C-4 position
exert a significant effect on the AChE, BuChE, b-amyloid and BACE-1
inhibitory profiles. In this regard, a C-2 amino-1-benzylpiperidine
was identified as a dual ChE binding pharmacophore whereas a C-
4 3,4-dimethoxybenzyl substituent was identified as a PAS binding
pharmacophore capable of inhibiting AChE-induced amyloid aggre-
gation. From our library, compound 7e [N2-(1-benzylpiperidin-4-
yl)-N4-(4-fluorobenzyl)pyrimidine-2,4-diamine] was identified as
the most potent dual ChE inhibitor (AChE IC50 = 7.7
lM; BuChE
IC50 = 2.2
l
M) whereas 7d [N2-(1-benzylpiperidin-4-yl)-N4-(4-bro-
mobenzyl)pyrimidine-2,4-diamine] exhibited potent b-amyloid
aggregation inhibition(AChE-induced = 53.9%; self-induced = 48.4%
at 100
dimethoxybenzyl)pyrimidine-2,4-diamine] was identified as
promising small molecule with multifunctional anti-AD profile
(IC50: hAChE = 9.9 M, eqBuChE = 11.4 M; hAChE-induced Ab40
M; self-induced Ab40-aggre-
M32–34 and BACE-1 = 34%
l
M). In addition, 7h [N2-(1-benzylpiperidin-4-yl)-N4-(3,4-
a
l
l
-
Figure 4. Superimposition of docked structures comparing the binding modes of
N2-(1-benzylpiperidin-4-yl)-N4-(3,4-dimethoxybenzyl)pyrimidine-2,4-diamine
(7h, Green) with that of donepezil (Red, 1, Fig. 1) in hAChE (PDB: 1B41).
aggregation inhibition = 59.3% at 100
l
gation inhibition = 17.4% at 100
l

4712
T. Mohamed et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4707–4712
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